RESUMO
BACKGROUND AND OBJECTIVE: Full-mouth scaling and root planing (FM-SRP) acts as a potent inflammatory stimulus immediately after treatment; however, systemic inflammation typically improves in the long term. The contribution of FM-SRP to systemic biological and acute-phase responses is largely unknown. The purpose of this prospective intervention study was to assess the systemic and local biological responses after FM-SRP. MATERIAL AND METHODS: Thirty-one patients with generalized moderate-to-severe chronic periodontitis received 1-stage FM-SRP. Measurement of clinical parameters and body temperature as well as collection of subgingival plaque, peripheral blood and gingival crevicular fluid was performed before and after treatment 2 or 3 times. Quantification of periodontopathic bacteria in the sulcus and measurement of corresponding serum IgG titers were performed. Systemic and local inflammatory markers such as endotoxin, high-sensitive C-reactive protein (hs-CRP) and 6 inflammatory cytokines were assessed using high-sensitivity assays. RESULTS: Compared to baseline values, FM-SRP resulted in a substantial improvement in clinical parameters (P < .05), lower bacterial counts (P < .01) and a significant decrease of IgG titers against Porphyromonas gingivalis (P < .001) 6 weeks after treatment. Comparing baseline parameters to those at 1 day post-treatment, there was a statistically significant elevation in body temperature (P = .007). In addition, a 5-fold increase in hs-CRP (P < .001), a remarkable increase in interferon-γ (P < .001) and a slight increase in interleukin (IL)-12p70 (P = .001) were detected in serum samples. In the gingival crevicular fluid, marked increases in hs-CRP (P < .001), IL-5 (P = .001), IL-6, IL-12p70 and tumor necrosis factor-α (P < .001 for the latter 3 markers) were noted 1 day after treatment. Endotoxin levels were below measurable limits for most time points. CONCLUSION: FM-SRP resulted in clinical and microbiological improvement 6 weeks post-treatment, but produced a moderate systemic acute-phase response including elevated inflammatory mediators 1 day post-treatment.
Assuntos
Periodontite Crônica/terapia , Raspagem Dentária , Mediadores da Inflamação/metabolismo , Aplainamento Radicular , Periodontite Crônica/microbiologia , Endotoxinas/sangue , Feminino , Seguimentos , Líquido do Sulco Gengival/química , Humanos , Imunoglobulina G/metabolismo , Japão , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Resultado do TratamentoRESUMO
UNLABELLED: An important goal for the improved diagnosis and management of infectious and inflammatory diseases, such as periodontitis, is the development of rapid and accurate technologies for the decentralized detection of bacterial pathogens. The aim of this prospective multicenter study was to evaluate the clinical use of a novel immunochromatographic device with monoclonal antibodies for the rapid point-of-care detection and semi-quantification of Porphyromonas gingivalis in subgingival plaque. Sixty-three patients with chronic periodontitis and 28 periodontally healthy volunteers were subjected to clinical and microbiological examinations. Subgingival plaque samples were analyzed for the presence of P. gingivalis using a novel immunochromatography based device DK13-PG-001, designed to detect the 40k-outer membrane protein of P. gingivalis, and compared with a PCR-Invader method. In the periodontitis group, a significant strong positive correlation in detection results was found between the test device score and the PCR-Invader method (Spearman rank correlation, r=0.737, p<0.0001). The sensitivity, specificity, and positive and negative predictive values of the test device were 96.2%, 91.8%, 90.4% and 96.7%, respectively. The detection threshold of the test device was determined to be approximately 10(4) (per two paper points). There were significant differences in the bacterial counts by the PCR-Invader method among groups with different ranges of device scores. With a cut-off value of ≥0.25 in device score, none of periodontally healthy volunteers were tested positive for the subgingival presence of P. gingivalis, whereas 76% (n=48) of periodontitis subjects were tested positive. There was a significant positive correlation between device scores for P. gingivalis and periodontal parameters including probing pocket depth and clinical attachment level (r=0.317 and 0.281, respectively, p<0.01). The results suggested that the DK13-PG-001 device kit can be effectively used for rapid, chair-side detection and semi-quantification of P. gingivalis in subgingival plaque. TRIAL REGISTRATION: UMIN Clinical Trials Registry (UMIN-CTR) UMIN000011943.
Assuntos
Técnicas Bacteriológicas/instrumentação , Cromatografia de Afinidade/instrumentação , Placa Dentária/microbiologia , Porphyromonas gingivalis/isolamento & purificação , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antibacterianos , Anticorpos Monoclonais , Técnicas Bacteriológicas/métodos , Cromatografia de Afinidade/métodos , Desenho de Equipamento , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Periodontite/microbiologia , Sistemas Automatizados de Assistência Junto ao Leito , Porphyromonas gingivalis/imunologia , Valor Preditivo dos Testes , Estudos ProspectivosRESUMO
BACKGROUND: Full-mouth scaling and root planing combined with azithromycin is clinically and bacteriologically effective for the treatment of chronic periodontitis. This study aimed to investigate the clinical and bacteriological effects of this combination treatment in patients with peri-implantitis. METHODS: Twenty adult patients with both chronic periodontitis and peri-implantitis were randomly divided into two groups (10: test, 10: control). All patients underwent full-mouth scaling and root planing but the test group received azithromycin for 3 days before the procedure. The probing depth, bleeding on probing, and the gingival index were assessed clinically. Bacterial samples were obtained before treatment at 1 week and 1, 3, 6, 9 and 12 months after treatment. Quantitative and qualitative analyses were performed using the polymerase chain reaction Invader method. RESULTS: All clinical parameters showed better improvement in both periodontitis and peri-implantitis in the test group. Periodontal bacteria were more effectively reduced in the test group, but gradually increased around implants 6 months after treatment and natural teeth 9 months after treatment. CONCLUSIONS: Full-mouth scaling and root planing combined with azithromycin was temporarily useful for the treatment of peri-implantitis. Clinical improvements were maintained for about 9 months but periodontal bacteria increased again 6 months after treatment.
Assuntos
Antibacterianos/uso terapêutico , Azitromicina/uso terapêutico , Raspagem Dentária/métodos , Peri-Implantite/tratamento farmacológico , Aplainamento Radicular/métodos , Idoso , Periodontite Crônica/tratamento farmacológico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Peri-Implantite/fisiopatologia , Índice PeriodontalRESUMO
Previous studies of oral microbiota by culture-dependent or targeted DNA approaches demonstrated that hyposalivation, a reduction in salivary secretions, might increase the amount of certain oral pathogens. However, the relationship between hyposalivation and the balance of oral microbiota, especially uncultivable bacteria, remains still unclear. The aim of this study was to elucidate the relationship between hyposalivation and oral microbiota by analyzing terminal restriction fragment length polymorphism (T-RFLP) of 16S rDNA. The 61 subjects were divided into two groups, hyposalivation group and normo-salivation group. The microbiota of tongue-coating samples was analyzed by T-RFLP. The amount of saliva, the number of Candida albicans, and also the dental status including plaque index, gingival index, bleeding on probing, probing pocket depth and decayed, missing, and filled teeth (DMFT) were assessed. Regarding the dental status, none of the evaluated factors were significantly different between the groups except the number of DMFT. According to the T-RFLP profiles, the patterns of microbiota in the tongue coating were classified into two groups, Clusters I and II. Cluster I is made up 76% of subjects with hyposalivation, while Cluster II is made up 61% of subjects with normo-salivation (p<0.001). Compared with the microbiota found in Cluster II, that in Cluster I had higher proportions of T-RFs corresponding to genera Veillonella, Dialister, Prevotella, Fusobacterium, and Streptococcus. T-RFLP analysis showed a significant role of salivary volume in determining the composition of the microbial community, regardless of the cultivability of the bacteria.
Assuntos
Fenômenos Fisiológicos Bacterianos , Biodiversidade , Microbiota/genética , Polimorfismo de Fragmento de Restrição , Xerostomia/microbiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Bactérias/genética , Candida albicans/fisiologia , DNA Ribossômico/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Componente Principal , Saliva/química , Saliva/microbiologia , Doenças Estomatognáticas/microbiologia , Língua/microbiologiaRESUMO
The rough lemon pathotype of Alternaria alternata produces host-selective ACR-toxin and causes Alternaria leaf spot disease of rough lemon (Citrus jambhiri). The structure of ACR-toxin I (MW = 496) consists of a polyketide with an α-dihydropyrone ring in a 19-carbon polyalcohol. Genes responsible for toxin production were localized to a 1.5-Mb chromosome in the genome of the rough lemon pathotype. Sequence analysis of this chromosome revealed an 8,338-bp open reading frame, ACRTS2, that was present only in the genomes of ACR-toxin-producing isolates. ACRTS2 is predicted to encode a putative polyketide synthase of 2,513 amino acids and belongs to the fungal reducing type I polyketide synthases. Typical polyketide functional domains were identified in the predicted amino acid sequence, including ß-ketoacyl synthase, acyl transferase, methyl transferase, dehydratase, ß-ketoreductase, and phosphopantetheine attachment site domains. Combined use of homologous recombination-mediated gene disruption and RNA silencing allowed examination of the functional role of multiple paralogs in ACR-toxin production. ACRTS2 was found to be essential for ACR-toxin production and pathogenicity of the rough lemon pathotype of A. alternata.
Assuntos
Alternaria/enzimologia , Alternaria/metabolismo , Citrus/microbiologia , Proteínas Fúngicas/metabolismo , Policetídeo Sintases/metabolismo , Alternaria/genética , Proteínas Fúngicas/genética , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Policetídeo Sintases/genéticaRESUMO
Osseous sarcoidosis is relatively uncommon, and treatment with corticosteroids is not always effective. Moreover, patients with an advanced stage of pulmonary sarcoidosis are sometimes infected with aspergillus in the cavities of the pulmonary lesions, and long-term use of corticosteroids should be prohibited to prevent the development of fatal invasive pulmonary aspergillosis. Here, we described a unique case of osseous sarcoidosis with pulmonary aspergillosis, showing a rapid improvement of the osseous symptoms just after the administration of the antifungal agent, itraconazole. Itraconazole is likely to become a candidate among new therapeutic agents for osseous sarcoidosis.
Assuntos
Antifúngicos/uso terapêutico , Doenças Ósseas/diagnóstico por imagem , Dedos , Itraconazol/uso terapêutico , Aspergilose Pulmonar/tratamento farmacológico , Indução de Remissão/métodos , Sarcoidose/diagnóstico por imagem , Doenças Ósseas/tratamento farmacológico , Diagnóstico Diferencial , Feminino , Seguimentos , Humanos , Pessoa de Meia-Idade , Aspergilose Pulmonar/complicações , Aspergilose Pulmonar/diagnóstico , Sarcoidose/tratamento farmacológico , Tomografia Computadorizada por Raios XRESUMO
BACKGROUND: Cumulative interceptive supportive therapy (CIST) is currently used as a guideline for treating peri-implant diseases. The objectives of this study were to determine the detection rate and measure the number of periodontopathic bacteria in lesions of different CIST levels and thereby characterize peri-implant disease from a bacteriological viewpoint. METHODS: This study included 105 patients who had both residual natural teeth and implants with peri-implant disease. A total of 105 implants were divided into levels A, B, C and D according to the CIST classification. Bacterial samples were collected from peri-implant pockets and four periodontopathic bacteria were measured by PCR and PCR-Invader assay. RESULTS: The number of periodontopathic bacteria increased in line with CIST level, and the detection rate was also associated with CIST level. However, no difference was found in the bacterial detection rate of P. gingivalis and T. denticola between CIST-B and CIST-C. There was a higher detection rate of all periodontopathic bacteria for CIST-D. CONCLUSIONS: The number of periodontopathic bacteria and detection rate increased as peri-implant disease advanced. However, there were no major differences in the detection rate between CIST-B and CIST-C. On the other hand, a higher detection rate of periodontopathic bacteria was seen for CIST-D.
Assuntos
Implantes Dentários/microbiologia , Bactérias Gram-Negativas/classificação , Peri-Implantite/microbiologia , Estomatite/microbiologia , Aggregatibacter actinomycetemcomitans/isolamento & purificação , Carga Bacteriana , Bacteroides/isolamento & purificação , Estudos Transversais , Placa Dentária/microbiologia , Feminino , Hemorragia Gengival/microbiologia , Humanos , Masculino , Pessoa de Meia-Idade , Peri-Implantite/classificação , Bolsa Periodontal/classificação , Bolsa Periodontal/microbiologia , Reação em Cadeia da Polimerase , Porphyromonas gingivalis/isolamento & purificação , Estomatite/classificação , Dente/microbiologia , Treponema denticola/isolamento & purificaçãoRESUMO
Mycobacteria are among the most common causes of hypersensitivity pneumonitis (HP), but controversy persists with regard to the involvement of the infectious potency of the organism in mycobacterial HP (hot tub lung). This study aimed to establish a mouse model of hot tub lung to clarify its pathophysiology. Mice were exposed intranasally to formalin-killed Mycobacterium avium from a patient with hot tub lung (HP strain) or chronic pulmonary infection (non-HP strain), and bronchoalveolar lavage fluids and lung tissues were evaluated for allergic inflammation. Dead M. avium HP strain, but not non-HP strain, elicited marked HP-like pulmonary inflammation in wild-type mice. Although the inflammation was induced in mice lacking CD4 or CD8, the induction of HP-like responses was prevented in mice lacking myeloid differentiation factor (MyD)88 or Toll-like receptor (TLR)9. Cultured lung CD11c+ cells responded to M. avium in a TLR9-dependent manner, and reconstitution of TLR9-/- mice with lung CD11c+ cells from wild-type mice restored the inflammatory responses. Further investigation revealed that pulmonary exposure to M. avium HP strain increased the number of lung CD11b+ CD11c+ cells (dendritic cells) through TLR9 signalling. Our results provide evidence that hot tub lung develops via the mycobacterial engagement of TLR9-MyD88 signalling in lung CD11b+ dendritic cells independent of the mycobacterial infectious capacity.
Assuntos
Alveolite Alérgica Extrínseca/metabolismo , Alveolite Alérgica Extrínseca/microbiologia , Antígeno CD11b/biossíntese , Antígeno CD11c/biossíntese , Mycobacterium/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Receptor Toll-Like 9/metabolismo , Idoso , Animais , Feminino , Humanos , Imunidade Inata , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium avium/metabolismo , Transdução de SinaisRESUMO
BACKGROUND AND OBJECTIVE: Enamel sheath protein (ESP) is involved in the construction of the enamel sheath during tooth development. The 17 kDa ESP is a one-step cleavage product processed by proteolysis from the N-terminal side of sheathlin (ameloblastin/amelin), one of the porcine enamel matrix proteins. Enamel sheath protein exhibits periodontal ligament and cementum regeneration activity in a buccal dehiscence model in dogs, and promotes the cytodifferentiation of cultured human periodontal ligament (HPDL) cells. The aim of this study was to determine the peptide segment on the C-terminal side sequence of the human ESP that possesses a cytodifferentiation activity on cultured HPDL cells. MATERIAL AND METHODS: The peptides synthesized on the basis of human ESP C-terminal side sequence were tested for their ability to increase the alkaline phosphatase (ALP) and mineralization activity of cultured HPDL cells. The expressions of osteocalcin, osteopontin and bone sialoprotein were measured by semi-quantitative PCR and therefore were determined to be specific indicators of mineralized tissue differentiation. RESULTS: Multiple synthetic peptides from the human ESP increased the ALP activity and stimulated matrix mineralization in long-term cultures of HPDL cells. Semi-quantitative PCR demonstrated the osteocalcin, osteopontin and bone sialoprotein expressions to increase relative to the control values. The peptide SDKPPKPELPGVDF had the strongest cytodifferentiation activity among all the synthetic peptides tested. CONCLUSION: A specific peptide sequence derived from the C-terminal side of the human ESP promotes the cytodifferentiation and mineralization activity of HPDL cells in a cell culture system.
Assuntos
Proteínas do Esmalte Dentário/síntese química , Proteínas do Esmalte Dentário/fisiologia , Ligamento Periodontal/citologia , Ligamento Periodontal/efeitos dos fármacos , Fosfatase Alcalina/biossíntese , Sequência de Aminoácidos , Animais , Calcificação Fisiológica/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Cementogênese/efeitos dos fármacos , Cementogênese/fisiologia , Proteínas do Esmalte Dentário/química , Proteínas do Esmalte Dentário/farmacologia , Humanos , Sialoproteína de Ligação à Integrina/biossíntese , Camundongos , Dados de Sequência Molecular , Osteocalcina/biossíntese , Osteopontina/biossíntese , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/farmacologia , Ligamento Periodontal/metabolismo , Regeneração/efeitos dos fármacos , Regeneração/fisiologiaRESUMO
The tangerine pathotype of Alternaria alternata produces host-selective ACT-toxin and causes Alternaria brown spot disease of tangerine and tangerine hybrids. Sequence analysis of a genomic BAC clone identified part of the ACT-toxin TOX (ACTT) gene cluster, and knockout experiments have implicated several open reading frames (ORF) contained within the cluster in the biosynthesis of ACT-toxin. One of the ORF, designated ACTTS3, encoding a putative polyketide synthase, was isolated by rapid amplification of cDNA ends and genomic/reverse transcription-polymerase chain reactions using the specific primers designed from the BAC sequences. The 7,374-bp ORF encodes a polyketide synthase with putative beta-ketoacyl synthase, acyltransferase, methyltransferase, beta-ketoacyl reductase, and phosphopantetheine attachment site domains. Genomic Southern blots demonstrated that ACTTS3 is present on the smallest chromosome in the tangerine pathotype of A. alternata, and the presence of ACTTS3 is highly correlated with ACT-toxin production and pathogenicity. Targeted gene disruption of two copies of ACTTS3 led to a complete loss of ACT-toxin production and pathogenicity. These results indicate that ACTTS3 is an essential gene for ACT-toxin biosynthesis in the tangerine pathotype of A. alternata and is required for pathogenicity of this fungus.
Assuntos
Alternaria/genética , Alternaria/metabolismo , Citrus/microbiologia , Micotoxinas/metabolismo , Policetídeo Sintases/metabolismo , Alternaria/classificação , Alternaria/patogenicidade , Regulação Fúngica da Expressão Gênica/fisiologia , Dados de Sequência Molecular , Estrutura Molecular , Micotoxinas/química , Micotoxinas/genética , Doenças das Plantas/microbiologia , Policetídeo Sintases/genéticaRESUMO
Mmp-20 and Klk4 are the two key enamel proteases. Can both enzymes process amelogenin to generate the major cleavage products that accumulate during the secretory stage of amelogenesis? We isolated Mmp-20 and Klk4 from developing pig teeth and used them to digest the tyrosine-rich amelogenin polypeptide (TRAP), the leucine-rich amelogenin protein (LRAP), and 5 fluorescence peptides. We characterized the digestion products by LC-MSMS, SDS-PAGE, and C18 RP-HPLC monitored with fluorescence and UV detectors. Mmp-20 cleaves amelogenin sequences after Pro(162), Ser(148), His(62), Ala(63), and Trp(45). These cleavages generate all of the major cleavage products that accumulate in porcine secretory-stage enamel: the 23-kDa, 20-kDa, 13-kDa, 11-kDa, and 6-kDa (TRAP) amelogenins. Mmp-20 cleaves LRAP after Pro(45) and Pro(40), producing the two LRAP products previously identified in tooth extracts. Among these key cleavage sites, Klk4 was able to cleave only after His(62). We propose that Mmp-20 alone processes amelogenin during the secretory stage.
Assuntos
Amelogenina/metabolismo , Calicreínas/metabolismo , Metaloproteinase 20 da Matriz/metabolismo , Alanina/metabolismo , Amelogênese/fisiologia , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Esmalte Dentário/metabolismo , Proteínas do Esmalte Dentário/análise , Eletroforese em Gel de Poliacrilamida , Histidina/metabolismo , Prolina/metabolismo , Serina/metabolismo , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Suínos , Espectrometria de Massas em Tandem , Triptofano/metabolismoRESUMO
BACKGROUND: To assess the efficacy and safety of S-1 and cisplatin with concurrent thoracic radiation for unresectable stage III non-small-cell lung cancer (NSCLC). METHODS: Eligible patients were 20-74 years old and had histologically or cytologically confirmed NSCLC, a performance status of 0-1, and no prior chemotherapy. Patients were treated with cisplatin (60 mg m(-2) on day 1) and S-1 (orally at 40 mg m(-2) per dose, b.i.d., on days 1-14), with the treatment repeated every 4 weeks for four cycles. Beginning on day 2, a 60-Gy thoracic radiation dose was delivered in 30 fractions. RESULTS: Of 50 patients, 48 were eligible. Partial response was observed in 42 patients (87.5%; 95% CI: 79.1-96.9%). This regimen was well tolerated. Common toxicities included grade 3/4 neutropenia (32%), grade 3/4 leukopenia (32%), grade 3/4 thrombocytopenia (4%), grade 3 febrile neutropenia (6%), grade 3 oesophagitis (10%), and grade 3 pneumonitis (5%). Median progression-free survival was 12.0 months and median overall survival was 33.1 months. The 1- and 2-year survival rates were 89.5 and 56%, respectively. CONCLUSION: This chemotherapy regimen with concomitant radiotherapy is a promising treatment for locally advanced NSCLC because of its high response rates, good survival rates, and mild toxicities.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/radioterapia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/radioterapia , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Carcinoma Pulmonar de Células não Pequenas/patologia , Cisplatino/administração & dosagem , Cisplatino/efeitos adversos , Terapia Combinada , Intervalo Livre de Doença , Combinação de Medicamentos , Feminino , Humanos , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Ácido Oxônico/administração & dosagem , Ácido Oxônico/efeitos adversos , Taxa de Sobrevida , Tegafur/administração & dosagem , Tegafur/efeitos adversosRESUMO
BACKGROUND: Non-tuberculous mycobacterial lung disease, most commonly caused by Mycobacterium avium infection, tends to show variable disease progression, and significant disease predictors have not been adequately established. METHODS: Variable numbers of tandem repeats (VNTR) were evaluated in 16 mycobacterial interspersed repetitive unit (MIRU) loci from M avium isolates cultured from respiratory specimens obtained from 2005 to 2007. Specifically, the association between VNTR profiles and disease progression was assessed. RESULTS: Among the 37 subjects who provided positive respiratory cultures for M avium during the 2005-6 period, 15 subjects were treated within 10 months following a microbiological diagnosis of progressive M avium lung disease. Nine subjects underwent long-term follow-up (>24 months) without treatment for stable M avium lung disease. Based on a neighbour-joining cluster analysis used to classify M avium-positive subjects according to the VNTR profile, subjects with progressive versus stable lung disease were found to be grouped together in distinct clusters. Further analysis using logistic regression modelling showed that disease progression was significantly associated with the genetic distance of the M avium isolate from an appropriately selected reference (age-adjusted odds ratio 1.95; 95% confidence interval 1.16 to 3.30; p = 0.01 for the most significant model). A best-fit model could be used to predict the progression of M avium lung disease when subjects from the 2005-6 period were combined with those from 2007 (p = 0.003). CONCLUSION: Progressive lung disease due to M avium infection is associated with specific VNTR genotypes of M avium.
Assuntos
Pneumopatias/genética , Infecção por Mycobacterium avium-intracellulare/genética , Mycobacterium avium/genética , Adulto , Idoso , Técnicas de Tipagem Bacteriana , Progressão da Doença , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Sequências de Repetição em TandemRESUMO
The tangerine pathotype of Alternaria alternata produces host-selective ACT-toxin and causes Alternaria brown spot disease. Sequence analysis of a genomic cosmid clone identified a part of the ACTT gene cluster and implicated two genes, ACTT5 encoding an acyl-CoA synthetase and ACTT6 encoding an enoyl-CoA hydratase, in the biosynthesis of ACT-toxin. Genomic Southern blots demonstrated that both genes were present in tangerine pathotype isolates producing ACT-toxin and also in Japanese pear pathotype isolates producing AK-toxin and strawberry pathotype isolates producing AF-toxin. ACT-, AK-, and AF-toxins from these three pathotypes share a common 9,10-epoxy-8-hydroxy-9-methyl-decatrienoic acid moiety. Targeted gene disruption of two copies of ACTT5 significantly reduced ACT-toxin production and virulence. Targeted gene disruption of two copies of ACTT6 led to complete loss of ACT-toxin production and pathogenicity and a putative decatrienoic acid intermediate in ACT-toxin biosynthesis accumulated in mycelial mats. These results indicate that ACTT5 and ACTT6 are essential genes in ACT-toxin biosynthesis in the tangerine pathotype of A. alternata and both are required for full virulence of this fungus.
Assuntos
Alternaria/genética , Coenzima A Ligases/genética , Enoil-CoA Hidratase/genética , Micotoxinas/biossíntese , Alternaria/enzimologia , Alternaria/patogenicidade , Citrus/microbiologia , Genes Fúngicos , Genômica , Interações Hospedeiro-Patógeno/genética , VirulênciaRESUMO
Alternaria brown spot, caused by the tangerine pathotype of Alternaria alternata, is a serious disease of commercially important tangerines and their hybrids. The pathogen produces host-selective ACT toxin, and several genes (named ACTT) responsible for ACT-toxin biosynthesis have been identified. These genes have many paralogs, which are clustered on a small, conditionally dispensable chromosome, making it difficult to disrupt entire functional copies of ACTT genes using homologous recombination-mediated gene disruption. To overcome this problem, we attempted to use RNA silencing, which has never been employed in Alternaria spp., to knock down the functional copies of one ACTT gene with a single silencing event. ACTT2, which encodes a putative hydrolase and is present in multiple copies in the genome, was silenced by transforming the fungus with a plasmid construct expressing hairpin ACTT2 RNAs. The ACTT2 RNA-silenced transformant (S-7-24-2) completely lost ACTT2 transcripts and ACT-toxin production as well as pathogenicity. These results indicated that RNA silencing may be a useful technique for studying the role of ACTT genes responsible for host-selective toxin biosynthesis in A. alternata. Further, this technique may be broadly applicable to the analysis of many genes present in multiple copies in fungal genomes that are difficult to analyze using recombination-mediated knockdowns.
Assuntos
Alternaria/genética , Citrus/microbiologia , Proteínas Fúngicas/genética , Micotoxinas/genética , Interferência de RNA , Alternaria/metabolismo , Alternaria/patogenicidade , Dosagem de Genes , Técnicas de Silenciamento de Genes/métodos , Genes Fúngicos , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Micotoxinas/biossíntese , Plasmídeos , RNA Fúngico/genética , Transformação GenéticaRESUMO
BACKGROUND AND OBJECTIVE: We have previously shown that increases in neutrophil elastase in periodontal ligament with chronic periodontitis results in degradation of the noncollagenous components. The purpose of this study was to investigate whether the destruction of noncollagenous components by treatment with elastase in vitro causes changes in the mechanical properties of the periodontal ligament. MATERIAL AND METHODS: The transverse sections of mandibular first molars, prepared from male Wistar rats at 6 wk of age, were digested with 0-50 microg/mL of neutrophil elastase at 37 degrees C for 4 h. Then, their mechanical properties and morphological features were examined. RESULTS: Digestion with elastase dose-dependently decreased the maximum shear stress and failure strain energy density of the periodontal ligament (p < 0.05-0.01). The histological observations after digestion revealed marked degradation of oxytalan fibers, but no marked changes of the collagen fibers, which was confirmed by the detection of very low quantities of hydroxyproline in the digest. The light and scanning electron micrographs showed that the elastase degraded the interfibrillar substances in the periodontal ligament and exposed individual collagen fibrils. CONCLUSION: These results suggest that the increased neutrophil elastase observed in periodontal disease degrades the oxytalan fibers and interfibrillar substances in the periodontal ligament to decrease its mechanical strength.
Assuntos
Elastase de Leucócito/metabolismo , Dente Molar/enzimologia , Ligamento Periodontal/enzimologia , Animais , Humanos , Elastase de Leucócito/administração & dosagem , Masculino , Dente Molar/diagnóstico por imagem , Ligamento Periodontal/química , Ligamento Periodontal/ultraestrutura , Radiografia , Ratos , Ratos Wistar , Resistência ao CisalhamentoRESUMO
In developing porcine enamel, the space between enamel rods selectively binds lectins and ameloblastin (Ambn) N-terminal antibodies. We tested the hypothesis that ameloblastin N-terminal cleavage products are glycosylated. Assorted Ambn cleavage products showed positive lectin staining by peanut agglutinin (PNA), Maclura pomifera agglutinin (MPA), and Limulus polyphemus agglutinin (LPA), suggesting the presence of an O-linked glycosylation containing galactose (Gal), N-acetylgalactosamine (GalNAc), and sialic acid. Edman sequencing of the lectin-positive bands gave the Ambn N-terminal sequence: VPAFPRQPGTXGVASLXLE. The blank cycles for Pro(11) and Ser(17) confirmed that these residues are hydroxylated and phosphorylated, respectively. The O-glycosylation site was determined by Edman sequencing of pronase-digested Ambn, which gave HPPPLPXQPS, indicating that Ser(86) is the site of the O-linked glycosylation. This modification is within the 15-amino-acid segment (73-YEYSLPVHPPPLPSQ-87) deleted by splicing in the mRNA encoding the 380-amino-acid Ambn isoform. We conclude that only the N-terminal Ambn products derived from the 395-Ambn isoform are glycosylated.
Assuntos
Proteínas do Esmalte Dentário/genética , Proteínas do Esmalte Dentário/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Glicosilação , Lectinas/metabolismo , Fragmentos de Peptídeos/metabolismo , Isoformas de Proteínas , Processamento de Proteína Pós-Traducional , Splicing de RNA , SuínosRESUMO
BACKGROUND AND OBJECTIVE: It has been reported that noncollagenous proteins may provide mechanical strength to the periodontal ligament. Several proteolytic activities, including that of neutrophil elastase, are reported to increase significantly in periodontal disease. The aim of this study was to investigate the function of neutrophil elastase in the initial destruction of periodontal ligament at early stages of periodontal disease. MATERIAL AND METHODS: The detection and identification of proteinases in chronic periodontitis and healthy periodontal ligament were examined by zymographic and zymo-Western analysis. The morphological changes of periodontal ligament, digested with or without authentic proteinases, were observed using scanning electron microscopy. RESULTS: Increases in neutrophil elastase, plasminogen, and matrix metalloproteinase-9 were detected in periodontal ligament from chronic periodontitis, compared with healthy periodontal ligament. Among these proteinases, only neutrophil elastase digested the intact noncollagenous proteins of periodontium. When human healthy periodontal ligament was directly digested by neutrophil elastase in an in vitro system, the morphological features were quite similar to that of the periodontal ligament in chronic periodontitis . In healthy periodontal ligament, the collagen fibrils are covered with noncollagenous proteins containing 110 kDa acidic glycoprotein, which was degraded initially by the neutrophil elastase. CONCLUSION: It was concluded that neutrophil elastase is involved in the degradation of noncollagenous protein-covered collagen fibrils in the early destructive stages of periodontal disease.
Assuntos
Elastase de Leucócito/metabolismo , Ligamento Periodontal/enzimologia , Periodontite/enzimologia , Adulto , Estudos de Casos e Controles , Doença Crônica , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Elastase de Leucócito/análise , Masculino , Metaloproteinase 9 da Matriz/análise , Proteínas Nucleares , Ligamento Periodontal/ultraestrutura , Plasminogênio/análise , Proteínas de Ligação a RNA , Receptores de Superfície CelularRESUMO
BACKGROUND AND OBJECTIVE: Commercially available enamel proteins, such as Emdogain, are clinically used for periodontal regeneration. However, the real mechanisms behind the bioactivities of enamel proteins is still unclear, as enamel proteins have multicomponents. The purpose of this in vivo study was to identify the cementum regeneration-promoting factor in enamel proteins that is clinically used for periodontal regeneration to induce cementum-promotive and osteopromotive activities. MATERIAL AND METHODS: Cementum regeneration, which is an important part of periodontal regeneration, was examined in experimental cavities prepared on a buccal dehiscence model of dogs. The purification of enamel protein with cementum regeneration activity was carried out by gel filtration and ion exchange chromatographies of newly formed secretory enamel. RESULTS: Cementum regeneration activity was found in the aggregate comprising 13-17-kDa sheath proteins along with a small amount of amelogenins, found in the newly formed secretory enamel. In these proteins, cementum regeneration activity was detected upon application of the 17-kDa sheath protein, but not by other lower molecular-weight sheath proteins and amelogenins. However, the purified 17-kDa sheath protein induced cementum regeneration activity only in a small area, although the regenerated cementum was thick. The activity of the 17-kDa sheath protein was believed not to have been a result of contamination by growth factors such as transforming growth factor-beta1 (TGF-beta1) found in the enamel protein, as the application of TGF-beta1 induced weak cementum regeneration activity. CONCLUSION: It is concluded that the 17-kDa sheath protein itself exhibits cementum regeneration activity, although other factors may be needed to demonstrate its full ability.
Assuntos
Cementogênese/efeitos dos fármacos , Cemento Dentário/efeitos dos fármacos , Proteínas do Esmalte Dentário/farmacologia , Perda do Osso Alveolar/patologia , Perda do Osso Alveolar/cirurgia , Animais , Regeneração Óssea/efeitos dos fármacos , Cromatografia em Gel , Cromatografia por Troca Iônica , Colágeno , Cemento Dentário/patologia , Proteínas do Esmalte Dentário/isolamento & purificação , Modelos Animais de Doenças , Cães , Eletroforese em Gel de Poliacrilamida , Órgão do Esmalte/química , Regeneração Tecidual Guiada Periodontal/métodos , Masculino , Doenças Mandibulares/patologia , Doenças Mandibulares/cirurgia , Osteogênese/efeitos dos fármacos , Ligamento Periodontal/efeitos dos fármacos , Ligamento Periodontal/patologia , Regeneração/efeitos dos fármacos , SuínosRESUMO
It has been shown that Emdogain Gel (Emd-Gel) containing enamel matrix proteins promotes biomineralization, such as osteogenesis and cementogenesis, during the regeneration of periodontal tissues. However, the growth factors involved in these activities of Emd-Gel remain unclear. In this study, Emd-Gel was fractionated into 22 sub-fractions by size exclusion chromatography. The osteoinductive factors, TGF-beta and BMP, were examined by a specific luciferase reporter gene assay. In the unfractionated Emd-Gel, TGF-beta-like activity was detected, while BMP activity was not. In contrast, in the fractionated Emd-Gel samples, TGF-beta-like activity was detected from fractions 8 to 13, and BMP-like activity was detected from fractions 4 to 6. Also, it was confirmed that the BMP-like activity in Emd-Gel was inhibited by authentic TGF-beta1 and TGF-beta-like activity. These results indicate that Emd-Gel contains both TGF-beta- and BMP-like growth factors that contribute to the induction of biomineralization during periodontal regeneration.